刚地弓形虫过氧化物氧化还原酶基因重组真核表达质粒的构建及表达  

Construction and expression of recombinant eukaryotic expression vector for peroxiredoxin gene of Toxoplasma gondii

作　　者：郝海霞[1] 王海龙[1] 殷丽天[2] 孟晓丽[1] 申金雁[1] 殷国荣[1] 

机构地区：[1]山西医科大学寄生虫学教研室,太原030001 [2]山西医科大学生理学系细胞生理学省部共建教育部重点实验室,太原030001

出　　处：《中国生物制品学杂志》2012年第7期809-811,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81071374)

摘　　要：目的构建刚地弓形虫(Toxoplasma gondii)过氧化物氧化还原酶(Peroxiredoxin,Prx)基因重组真核表达质粒,并在HEK293T细胞中进行表达。方法以重组质粒pET-30a(+)/TgPrx为模板,PCR扩增TgPrx基因片段,插入真核表达载体p3×Flag-CMW-14,构建重组表达质粒p3×Flag-CMW-14/TgPrx,转染HEK293T细胞,RT-PCR和Western blot检测TgPrx基因的转录和蛋白的表达。结果 PCR扩增出591 bp的TgPrx基因片段;重组真核表达质粒p3×Flag-CMW-14/TgPrx经PCR、双酶切及测序证明构建正确;转染重组表达质粒的HEK293T细胞可检测到TgPrx基因的转录和蛋白的表达。结论成功构建了重组真核表达质粒p3×Flag-CMW-14/TgPrx,并在HEK293T细胞中正确表达,为进一步研制核酸疫苗奠定了基础。Objective To construct the recombinant eukaryotic expression vector for peroxiredoxin （Prx） gene of Toxoplasrna gondii （Tg） and express in HEK293T cells. Methods TgPrx gene was amplified by PCR using recombinant plasmid pET-30a （＋）/ TgPrx as a template, and inserted into eukaryotic expression vector p3× Flag-CMW-14. HEK293T cells were transfeeted with the constructed recombinant plasmid p3×Flag-CMW-14/TgPrx, and determined for transcription of TgPrx gene and expression of TgPrx protein by RT-PCR and Western blot respectively. Results TgPrx gene fragment at a length of 591 bp was amplified by PCR. PCR, restriction analysis and sequencing proved that recombinant plasmid p3 × Flag-CMW-14/TgPrx was constructed correctly. Both the transcription of TgPrx gene and expression of TgPrx protein were observed in HEK293T cells transfected with the constructed recombinant plasmid. Conclusion Recombinant plasmid p3 × Flag-CMW-14/TgPrx Was successfully constructed and expressed in HEK293T cells, which laid a foundation of preparation of DNA vaccine.

关 键 词：刚地弓形虫 过氧化物氧化还原酶 真核细胞 基因表达 

分 类 号：R382.5[医药卫生—医学寄生虫学] Q786[医药卫生—基础医学]